Project Summary Liver cancer is the 6th most common cancer and the 4th most common cause of cancer death in the world. According to CDC, the incidence rate of liver cancer has doubled over last 20 years, making it the fastest growing cancer in US. It is estimated that there will be 42,220 new cases and 30,200 death of liver cancer in 2018. Thus, we are addressing an increasing health problem in US. Although liver resection is curative, the lack of adjuvant therapy becomes a critical barrier to the success of surgery, resulting in ~70% 5yr recurrence rate. Thus, novel therapies are urgently needed and immunotherapy has a great potential. Majority (80-90%) of liver cancer is hepatocellular carcinoma (HCC), which re-express alpha fetoprotein (AFP). Engineering patient?s autologous T cells with AFP epitope-specific T cell receptor (TCR) genes offers a specific immunotherapy for HCC. Currently, one AFP158-specific high-affinity TCR just got approved for clinical trial in May of 2017. But, high-affinity TCRs may cause severe on- and off-target toxicity. We reason that it will require a panel of TCRs with different affinity to find the optimal TCRs that enable human T cells to kill HCC tumor cells without toxicity. Thus, we propose to identify multiple AFP-specific TCRs and test their antitumor efficacy and toxicity. We hypothesize that an effective in vivo immunization strategy will induce potent human AFP (hAFP)-specific CD8 response with diverse TCR repertoire in HLA-A2 mice. This large number of CD8 cells enables us to directly study their antitumor function and identify a panel of TCRs with different affinity, increasing the chance of finding the optimal TCRs to create TCR-Ts of potent antitumor effect without toxicity. To test this hypothesis, we have developed a concrete research strategy with three specific aims. A1: Identify hAFP158-specific TCRs to create TCR-Ts of different functional avidity; A2: Identify the hAFP158-specific TCRs without or with minimal cross-reactivity; and A3: Create expandable and removable TCR-Ts with stemness to achieve potent in vivo antitumor effect. The goals are to obtain the optimal TCR genes that can engineer primary human T cells to become effective TCR-Ts capable of killing human HCC cells without harming normal cells and to establish protocol for generating safe and effective TCR-Ts that are ready for clinical trials. In the end, applications for an investigative new drug and clinical trial will be filed. This will eventually lead to new therapeutic approach for HCC. In addition, the methodical approach of creating AFP-specific TCR library and identifying optimal TCRs with potent antitumor effect and no toxicity will serve as a platform for other antigens and HLA haplotypes, which will greatly impact the field of tumor immunotherapy.